The present invention relates generally to high pressure capillary liquid chromatography, and more particularly to a method and apparatus for high-pressure delivery of solvent or solvent mixtures, such as is useful in the art of liquid chromatography.
The practice of HPLC generally requires that a molecular species to be separated or analyzed be dissolved in a liquid, the mobile phase, and conveyed by that liquid through a stationary phase. In the stationary phase, a large surface area is presented which is in intimate contact with the mobile phase. Mixtures of analyte compounds, dissolved in the mobile phase, can be separated during passage through the column by processes of adsorption or retention, which act differently on the various analyte species. The differential retention causes the analytes to elute from the column with respect to time and volume. The eluting analytes will typically transit through an in-line detector, where quantitative and/or qualitative examination of analytes will occur.
High pressure liquid chromatography (HPLC) solvent delivery systems are used to source either single-component liquids or mixtures of liquids at selected pressures which can range from substantially atmospheric pressure to pressures on the order of ten thousand pounds per square inch. The above pressures are required to force the mobile phase through the fluid passageways of a stationary phase support, where separation of dissolved analytes can occur. The stationary phase support may comprise a packed bed of particles, a membrane or collection of membranes, a microfabricated structure typically comprising an array of fluid passageways etched into a solid support, or an open column or tube.
Often, analysis conditions require the mobile phase composition to change over the course of analysis which is termed xe2x80x9cgradient elutionxe2x80x9d. In gradient elution, the viscosity of the mobile phase may change and the pressure necessary to maintain the required volumetric flow rate will change accordingly.
The elution behavior of analyte molecules is a function of the characteristics of both the stationary and mobile phases. To the extent that the properties of the stationary phase may remain substantially fixed throughout the analysis, variation in elution behavior is predominantly a result of variation in the properties of the mobile phase. In an xe2x80x9cisocratic modexe2x80x9d of chromatography, the solvent composition remains substantially constant as a function of time, and analytes in the sample will tend to elute when a prescribed mobile phase volume has transited the column. In a xe2x80x9cgradient modexe2x80x9d of chromatography, the solvent composition is required to change as a function of time, tracking a user defined profile. In this mode, analytes will elute in response to both the composition and the volume of solvent delivered.
The separation process occurring in liquid chromatography can result in the separation of an injected sample mixture into its component parts. These component parts are eluted from the column in reasonably distinct zones or bands. As these bands pass through a detector, their presence can be monitored and a detector output can be produced. This output includes a pattern of analyte concentration within the eluting bands, which can be represented by means of a time varying electric signal, and gives rise to the nomenclature of a xe2x80x9cchromatography peak.xe2x80x9d These peaks may be characterized with respect to their retention time, that is, the time in which the center of the band transits the detector relative to the time of injection. In many applications, the retention time of a peak is used to infer the identity of the eluting analyte based upon related analyses incorporating standards or calibrants. The retention time of a peak is strongly influenced by the mobile phase composition and by the volume of mobile phase, which has passed over the stationary phase.
The utility of chromatography relies heavily on run-to-run reproducibility, such that a given analysis can be compared with an analysis of standards or calibrants with confidence in the resulting data. Known pumping systems exhibit some non-ideal characteristics which result in diminished separation performance and diminished run-to-run reproducibility.
Among the non-ideal pump characteristics exhibited in known pumping systems are, generally, fluctuations in solvent composition and/or fluctuations in volumetric flow rate. Such volumetric flow rate fluctuations in present and known HPLC pumping systems disadvantageously cause varying retention times for a given analyte. That is, the amount of time that an analyte is retained in the stationary phase fluctuates undesirably as a function of the undesirable volumetric flow rate fluctuations. This creates difficulties in inferring the identity of a sample from the retention behavior of the components. Volumetric flow rate fluctuations can result in fluctuations in solvent composition when the output of multiple pumps is summed to provide a solvent composition.
Fluctuations in solvent composition in present and known HPLC systems disadvantageously result in interactions with the systems analyte detector and produce perturbations which are detected as if they arose from the presence of a sample. In effect, an interfering signal is generated. This interfering signal is summed with the actual signal attributable to the analyte, producing errors when the quantity of an unknown sample is calculated from the area of the eluting sample peak.
In light of the above, the requirements imposed on HPLC solvent delivery systems are severe. HPLC pumps are typically required to deliver solvents at pressures which can range from several pounds per square inch to as much as 10,000 pounds per square inch. There are problems and non-ideal effects associated with delivering liquids for chromatography against elevated pressures including seal deformation under load and absolute seal leakage. HPLC pumps are expected to output the mobile phase solvent at precisely controlled flow rates in a smooth and uniform manner. In the case of gradient chromatography or in the case of isocratic chromatography, where a fixed solvent composition is blended in real time during the separation, there is the further requirement that mobile phase composition as well as flow rate be precisely and accurately controlled during delivery. However, system operating pressures may be changing very substantially during the separation and the compressibilities of the constituent mobile phase solvents may be quite different.
The large errors associated with the compression or relaxation of large volumes of fluid can be minimized by the use of small volume syringe pumps that utilize multiple syringe strokes to deliver solvent through the course of a chromatographic separation period. However, these pumps typically suffer from flow perturbations associated with the transition of fluid delivery from one syringe cycle to the next.
An emerging area of chromatographic separation and analysis is developing around the use of extremely narrow bore separation columns. Such columns have been termed xe2x80x9ccapillary columnsxe2x80x9d with diameters typically in the range of 30 to 800 micron internal diameter. Such columns may be packed with a particulate packing material, or in the smallest diametral range, the stationary phase may be provided by the column wall itself or a coating applied to that wall. Mobile phase flow rates for such particulate packed capillary columns can typically range from approximately one nanoliter per minute to ten or more microliters per minute. These figures represent a three to six order-of-magnitude reduction in flow rate and consequently a similar reduction in the volume of the separation from what is currently practiced on, for example, the four millimeter internal diameter columns widely commercially available. HPLC systems designed around capillary columns have particular utility when the HPLC separation is coupled with a downstream process that does not readily tolerate large amounts of HPLC mobile phase, or where the use of unusually expensive mobile phases is desired. Examples of such processes are: (1) mass spectrometry, which requires that the sample reside in the gas phase at high vacuum conditions prior to mass analysis, (2) infrared spectroscopy, where organic solvents used for HPLC must be eliminated because they represent an interference to analyte detection in the infrared region of the electromagnetic spectrum, (3) microfraction collection, which requires that the analyte be deposited in a small volume on a collection substrate with minimum associated background contamination from the HPLC mobile phase, and (4) nuclear magnetic resonance spectroscopy (NMR) which can benefit from significant signal background reduction through the use of somewhat exotic mobile phases, such as deuterium-substituted mobile phases in the case of proton-NMR.
Substantially, the same requirements for precision and accuracy of solvent composition and flow rate delivery exist as for larger scale chromatography, but the mechanisms for controlling delivery must function at approximately one one-thousandth or less of the conventional volume scale. In particular, the non-idealities of a given implementation which could be dismissed at a much larger volumetric scale give rise to overwhelmingly large perturbations to a system of the scale of capillary HPLC.
A known implementation of a continuous-delivery pumping system for normal analytical scale HPLC (1 to 4 mm column ID), available from Hewlett-Packard and selling under the product designation HP1090/DR5, formed a gradient at run time using a plurality of positive-displacement piston pumps operating against a modest intermediate pressure. The summed output was fed to a low-pressure accumulator and then to a serial or in-line booster pump stage constructed on a diaphragm principle. The booster pump operated at a fixed frequency of approximately 10 Hz, but had a variable stroke volume which accommodated the output flow of the solvent metering/proportioning stage. The booster stage would take the proportioned mixture from the intermediate pressure to the full system operating pressure, which was in the conventional (5000 to 6000 PSIG) HPLC range. Because of the series construction employed, the pump had a delay volume of not less than approximately 300 microliters. This delay volume is quite acceptable for 2 mm ID and 4mm ID column work, but becomes significant at 1mm ID column size, and is inappropriate for work in the capillary realm, particularly so when the capillaries extend down to 30 to 50 micron internal diameter, and the flow rates are in the nanoliters-per-minute region.
A further disadvantage of that implementation is that the diaphragm booster stage imparted a significant AC pressure pulsation in the range of 100 PSIG, at the 10 Hz stroke frequency, to the downstream system. The existence of this AC pulsation in turn required the use of a compliant pulse damper in continuity with the diaphragm pump output stream, which is not desirable from several standpoints. Finally, because the system was designed to perform continuous solvent delivery at flow rates up to 5 milliliters per minute, as required for 4 mm ID columns, each of the intermediate-pressure solvent pumps was itself a continuous-delivery reciprocating pump comprising two cylinders and associated valving, resulting in a complex and expensive system.
Davis, M. T., et al., J. Am Soc. Mass Spectrom., 6, 571-577 (1995) describe an alternate implementation of a solvent delivery system designed to be operable at a capillary size scale. The approach disclosed by Davis et al. involves forming gradients by employing two low-pressure syringe pumps to generate a solvent composition which temporarily resides in a loop of open tubing. This loop is connected at each end to a commercially available six-port switching valve, following the manner of an external loop injector. Upon actuation of the switching valve, the fluid contents of the external loop are placed in continuity with the column, and with a high-pressure-capable syringe pump. The syringe pump is operated as a pressure source to expel the fluid contents of the external loop through the capillary analytical column. In arguing for a constant-pressure mode of operation, the authors acknowledge that their system is substantially incapable of constant-flow operation, as xe2x80x9cThere is no direct measurement of the actual flow from the column, and no feedback to the pump to maintain that flow rate. The piston is simply moved at a constant rate, and it can take a surprisingly long time for a system to come to equilibrium, and measured flow rates can change considerably during the course of a run.xe2x80x9d The system which Davis et al. describe includes a very large-volume syringe, representing a significant hydraulic capacitance, connected to a relatively small-diameter, and therefore substantially restrictive, capillary column. Such a system would indeed be expected to suffer from a very long hydraulic time-constant, or flow-rate settling time, in response to either a resistance change of the analytical column, or a flow-rate change at the syringe pump. This settling time could range from many minutes to hours. Therefore, for all practical purposes, during the course of a chromatographic run, there would be little correlation expected between the syringe pump delivery flow rate and the column flow rate. The authors claim that xe2x80x9cthere is no significant difference between constant pressure and constant flow operation when peptides and proteins are separated by capillary HPLCxe2x80x9d, yet acknowledge that during constant pressure operation, there are flow rate changes arising from the changes in solvent viscosity during gradient elution, and that constant-pressure operation is sensitive to xe2x80x9cflow problems caused by sample precipitation or other blockage in the tubing or on the columnxe2x80x9d.
In fact, when samples drawn from biological matrices are separated by capillary chromatography, it is not unusual to see the column resistance change measurably from one run to the next. This effect is superimposed upon the column resistance variation typically observed from one column to another, which is attributable to minor variations in physical construction and packing. Even small flow rate changes impair the user""s ability to quantitate sample amounts from detector peak areas, since the area measurement assumes a constant flow rate. Larger flow rate changes impact system properties such as the ability to successfully maintain stable spray behavior in electrospray/nanospray interfaces. These examples illustrate why HPLC has been optimally practiced at constant volumetric flow rate as opposed to constant pressure. The Davis system provides the user with substantially no quantitative measure of the actual delivery flow rate provided to the column.
Finally, the approach disclosed by Davis et al. teaches the use of a loop of open tube as a gradient storage device. FIG. 3 in the Davis reference shows that there is only a very crude relationship between the called-for and the actual gradient composition profile delivered to the analytical column when a loop of open tube is used as the storage device. This behavior is not surprising in light of the fact that an open tube storage device will be susceptible to compositional band or zone broadening due to the Poiseuille flow profile attained in the open tube during fluid loading and expulsion, and to density-driven fluid motion while the solvent composition gradient is resident in the tube.
Recently, interest has developed in the ability to perform HPLC separations using extremely small diameter packing materials, that is, less than three micron diameter, with concomitantly high mobile phase pressure, which is required to drive the liquid through a bed filled with such packing particles. Enhanced separation characteristics of HPLC are demonstrated, in particular, either the absolute peak capacity of the separation, or the throughput of the separation as expressed in peaks eluted per unit time, can be substantially improved through the correct use of small packing particles and a high system pressure. The utility of particles as small as one micron diameter and system pressures in the rage of 10,000 to 100,000 PSIG has been demonstrated. However, in such analysis, power dissipation in the form of self-heating of the columns can be significant under the conditions of extremely high pressure drop over the length of the column. Therefore, it is desirable to maintain the column diameter as small as is feasible, often in the range of 30 to 75 micron internal diameter. These small column diameters, with correspondingly small volumetric flow rate requirements, help maintain the total heat generated to a relatively small value, and also keep the thermal paths for heat dissipation relatively short.
In using such small diameter columns (30 to 75 micron internal diameter), the actual volume of separation becomes exceedingly small, given that the mobile phase flow rates are typically in the 5 to 200 nanoliter per minute range. Gradient formation requires that the individual components of the mobile phase must be delivered at levels as low as 0.1% of the total system flow rate. In addition, high-pressure gradient formation pumps must deliver the component flows in a manner which is reproducible in fashion, against the full system operating pressure, without perturbations. The above described analytical requirements imply the ability to quantitatively deliver component flow rates as small as 10 picoliters per minute against pressures as high as 100,000 PSIG. Known HPLC systems do not provide the sealing and actuator technology required for the above stated requirements at a cost which is reasonable in the intended market.
Therefore, in light of the above, flow-splitting based systems have come under investigation for performing separations using extremely small packing particles with concomitantly high mobile phase pressure to drive the fluid through the bed of packing particles. These flow-splitting systems use solvent composition generation and pressure augmentation at a size scale of convenience. This larger scale flow is directed to a shunt path which includes significant resistance in line. The small scale gradient flow required for the above-stated 30 to 75 micron internal diameter columns is tapped from the larger scale flow by means of a tee and is subsequently conveyed through the injection means toward the column. Such systems have been employed extensively in capillary chromatography at modest pressures (0 to 5,000 PSIG) and their operation is being explored at more elevated pressures in the range of 6,000 to 20,000 PSIG. Drawbacks of these flow-splitting systems become apparent when system pressures reach the 50,000 to 100,000 PSIG levels, in part due to heat dissipation in the shunt path. In extreme cases, the heat generated is enough to convert the prescribed liquid into a vapor, or at a minimum, a liquid having a substantial vapor component.
Thus it should be apparent that the implementation of systems intended for use with extremely small packing particles and high mobile phase pressures involves severe design challenges.
The present invention provides an HPLC solvent delivery system which accomplishes low volume solvent composition formation at a selected pressure, for example, at substantially atmospheric pressure. Such low volume, low pressure solvent composition formation is followed by pressurization of the formed solvent composition, which is in turn followed by expulsion of the formed solvent composition at a higher pressure into a receiving device such as a column. The formation of the solvent composition at low pressure can also include the introduction of the sample analyte as a step or process included within the solvent composition formation.
According to the invention, a high pressure capillary liquid chromatography solvent delivery apparatus and method uses low volume solvent composition formation at a selected pressure, for example, at substantially atmospheric pressure, followed by pressurization of the formed solvent composition . The solvent composition formation is performed at a substantially low volume and a selected pressure, for example, at substantially atmospheric pressure, and is then pressurized. Expulsion of the formed solvent composition is then effected at this higher pressure into a receiving device such as a column. The solvent composition is formed from a solvent composition generator or fluid metering system which may be optimized to deliver liquids at low pressures into a receiving solvent composition storage matrix via an isolation valve. The solvent composition delivered to the storage matrix is in reverse order such that the first volume of the solvent composition delivered to the storage matrix is the last volume of the solvent composition expelled out. The solvent composition so formed can include sample analyte introduced via a low-pressure injector, the sample volume existing as a band or zone within the formed solvent composition . Pre-existing liquid within the storage matrix is displaced into a compliant, variable-volume fluid accumulator such as a pleated bellows or a diaphragm, which is in fluid communication with the storage matrix. The fluid accumulator is enveloped in a pressure caisson or chamber which can be pressurized or depressurized in response to sensor signals obtained from a fluid volume displacement transducer (or sensor) responsive to the volume change of the fluid accumulator, and in response to a pressure sensor in fluid communication with the caisson, in accordance with solvent composition cycle formation. Closure of the isolation valve isolates the solvent composition generator, and the low-pressure injector, if present, from the pressures developed during the solvent composition pressurization and expulsion phases of system operation. As the caisson is pressurized, the solvent composition within the storage matrix is expelled out past the isolation valve by the pressure acting on the fluid accumulator and its fluid contents. The solvent composition is then delivered to the chromatographic column via a path which can include a high-pressure capable injector.
Advantages of the present invention include operability at pressures above normal HPLC operating pressures, with the potential to utilize smaller chromatographic packing particles, in order to obtain enhanced separation characteristics of the chromatographic system, including improvements in the absolute capacity of the separation and/or the throughput of the separation as expressed in peaks eluted per unit time. The system and method are scaleable, and can be used with relatively more moderate pressures and column internal diameters. When employed with suitably small columns and volumetric flow rates, the total power dissipation resulting in heat generation within the column can be kept relatively small, and the associated thermal paths for heat dissipation can be relatively short.
Another advantage of the high pressure capillary liquid chromatography solvent delivery system is that it is operational at elevated pressures without the use of dynamic seals exposed to high pressure whose failure would give rise to compositional fluctuations or errors. Rather, the system may be constructed using a single dynamic seal at the pressure caisson, whose sealing performance is non-critical, in that, the actuator rate being applied to the piston which pressurizes the caisson is not used to infer the delivery flow rate.
In the gradient mode of chromatography, peak retention behavior is primarily dictated by solvent composition and according to the invention, the accuracy of maintaining the solvent composition profile is enhanced by the fact that this profile is generated under conditions of substantially constant and relatively low pressure, where the fluid metering behavior can be optimized. If syringe pumps are employed to perform this low-pressure fluid metering function, their behavior can be optimized because the low axial loads on the syringe pump pistons permit the use of drive screws with micrometer-quality threads, at relatively modest cost, and obviate the need for heavy thrust bearings which can degrade the accuracy of the syringe piston linear translation mechanism. The syringe pump seals are not subjected to high loads which would result in either excessive seal leakage or significant seal deformation. The fact that the fluid metering leading to solvent composition formation is accomplished at low pressure, enables the use of alternate fluid metering technologies, including those which are normally constrained to work at pressures near atmospheric pressure, an example being integrated microfluidic dispensing or delivery systems which utilize one or more diaphragm-style pumping elements typically actuated by solenoids or piezo-electric actuators.
Because the short-term flow rate through the chromatographic column is governed by caisson pressure, which is monitored and controlled via feedback loop, the requirements for translational accuracy imposed on the lead screw or jack mechanism actuating the caisson piston are relatively simple. This is in contrast to lead screw requirements which exist for high-pressure solvent composition generation mechanisms used in current capillary liquid chromatography systems.
A further advantage of the presently disclosed chromatography system and methodology includes features drawn to stopped-flow spectroscopic scanning. The system allows for the solvent liquid flow, and therefore chromatographic peak elution, to be intermittently stopped, whereupon the caisson is rapidly depressurized and repressurized under pressure servo control without substantially varying the compositional attributes of the emerging solvent composition.
An additional advantage of the present invention derives from employing a series pump architecture in a manner that minimizes delay volume associated with the pressure boosting stage of the solvent delivery process. The formed solvent composition flows off the storage matrix directly into the analytical column, being displaced from the storage matrix by the re-introduction of fluid previously resident within the accumulator. Moreover, the solvent compositions in the present invention can be formed at a flow rate convenient to the protocol being employed. Typically, the solvent composition formation flow rate is multiple (many) microliters per minute, while the expulsion flow rate may be only a few nanoliters per minute. These two processes are decoupled in time, allowing the fluid metering elements to perform in a flow regime where their behavior is more nearly ideal.
Another feature attendant to the present invention is that only a single dynamic seal is maintained in the pressurized portion of the system. Additionally, its leakage characteristics, short of outright seal failure, are substantially decoupled from the chromatographic delivery, in that both the caisson pressure, and the volumetric delivery during fluid transfers into and out of the storage matrix, are independently measured. Further, the fluid accumulator allows fluid displacement to occur into or out of the storage matrix while implementing a hermetic-quality barrier between the working fluid within the caisson and the chromatographic stream.
Still another advantage to the present invention is the ability to generate and store the solvent compositions for an entire chromatographic run, due to the diminutive scale of capillary chromatography, and therefore, it is not of necessity to have continuous-flow metering pumps to form the solvent compositions at run-time as is true in other commercial systems. This contributes to the overall simplicity of the current chromatography system.
The herein described high pressure capillary liquid chromatography solvent delivery system and methodology is capable of providing reasonable solvent delivery operation at both low and high pressure extremes which are unattainable with present HPLC systems. The present invention is particularly well-suited for systems directed to high peak capacity separations employed in coupled liquid chromatography-mass spectrometry (LC-MS) and the like.